Leishman001

Oh yeah, condensation on the lids can be controlled a bit by stacking the plates in one "tower" after pouring and leaving them at room temperature over night. Repackage them in the morning and store at 4 C.

Hi:

There have been several very good answers to your questions. Here's my two cents.

I prepare LB-agar in 500 ml batches in Kimax bottles and autoclave to sterilize. I put stir bars in the bottles before autoclaving (make sure the bottle bottom is flat or the bar will not stir properly). 500 ml of LB-agar is enough to be poured into a standard sleeve of 25 petri plates and takes about 1 hour to cool down to where you can add antibiotics and pour. I stir the agar on a stir plate during the cooling process to avoid localized cooling in the bottle, avoiding "chunks" of agar when pouring.

To test if the agar is cool enough to add antibiotics without cooking them, I CAREFULLY touch the bottle to the inner skin of my forearm (wait at LEAST 45 minutes before attempting this to avoid burns). If I can hold the bottle there for 5-10 seconds, the agar is probably cool enough to pour. I add the antibiotics, give the agar a quick final stir and pour. To prevent the stir bar in the bottle from flopping around, I use a second magnet on the outside of the bottle to hold the first in place.

If I want to make batches to cool to store, I make and sterilize the LB-agar the same way and put it in a quiet place with no traffic to cool. Air currents generated by moving people increase the chance of a contaminant getting under the cap and ruining your agar. Keep the cap loose during the cooling process to avoid a vacuum but cap it once the agar is mostly cool. I usually store the agar at room temp for about a month.

To re-liquify the agar, we have access to a steam chamber (not an autoclave, although that can also be used). This melts the agar in about 1-1.5 hours and avoids the spurting caused by microwaving the solid agar.

To answer your first question, you can put the molten agar at 55-60 C to store it for a few hours. If you incubate at this temperature for longer, the sugars in the agar will start to carmelize and the agar will turn dark and eventually appear almost black. Don't use it once it starts to change colour.

Hello Oana:

Try this website out for answers to your question:
http://www.protocol-...sts/15937.html.
Someone else asked the same question on a different forum.

MQ-grade water can be deemed to be more "pure" in terms of dissolved ions than distilled water, where ions dissolved in the water before distillation can be carried forward into the end product, even after double-distillation.

MQ-grade water has become the industry standard, but distilled was the norm when I started my career. Just because your protocol calls for MQ water now doesn't mean you can't substitute in double-distilled. Make sure you sterilize the final products (TC media, transformation mix) before you use them.

Good luck!